In body fluids, there exist several physiologically important biochemical systems that act through activation and subsequent inactivation of proteolytic enzymes, such as, in blood, the coagulation system, the fibrinolytic system and the complement system, and in gastro-intestinal juices the digestive enzymes. For the assessment of biological function of these systems it is important to be able to follow the course of such proteolytic activity in time. Such function assessment is of paramount diagnostic importance because disturbances of such systems can lead to fatal diseases like coronary infarction, stroke or fatal bleeding (blood coagulation and fibrinolysis), generalised infections and autoimmune diseases (complement system) or disturbed adsorption of food (gastrointestinal juices).
In methods of determining the thrombin generation in plasma or platelet-rich plasma according to the state of the art, the plasma turns into a gel after several minutes. This is due to the conversion of fibrinogen into insoluble fibrin. This fibrin polymerizes, so that a fibrin-network is formed which, in fact, is the actual clot. This clot is similar to a sponge that contains fluid (i.e. plasma or serum) in which thrombin is generated. During this generation of thrombin the signal substrate is converted into a signal-producing leaving group. Since the gel is relatively transparent it is possible to record the fluorescent signal in time in clotting plasma. However, in case of measurement in whole blood the amount of signal is considerably reduced (typically less than 5% of the amount measured in clear plasma) and any movement of the erythrocytes greatly influences the signal. The fibrin network (the gel) that is formed captures the red blood cells and after a while “clot retraction” occurs that results in inhomogeneity of the gel thereby separating fluid captured inside the gel from fluid that is present outside the fibrin network. Following the picture of the “spongy network”, clot retraction results in a situation that is similar to part of the sponge being squeezed out. This has such an influence on the signal that measurement would be almost impossible.
WO 03/093831 describes a suitable technique to measure the activity of thrombin in real time in plasma or platelet-rich plasma. The technique includes the addition of a signal substrate to said biological medium. The proteolytic enzyme is able to convert the substrate, and the leaving group of the substrate can be measured with an appropriate technique. This can be the measurement of fluorescence, optical density, NMR, and the like, the choice of which mainly depends on the nature of the signal. When fluorescence is used, then it is in principle possible to measure in turbid solutions such as platelet-rich plasma or plasma that contains fibrin. Also measurement in whole blood, i.e., plasma containing platelets, white blood cells and erythrocytes, would be possible. However, the presence of red blood cells has a great disturbing impact on the signal.
Therefore, there is still a need for a method for measuring the thrombin generation in real time in whole blood in a reliable and simple manner. The present invention provides such a method.